EP1753868A2 - Production de produits chimiques fins par fermentation - Google Patents

Production de produits chimiques fins par fermentation

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Publication number
EP1753868A2
EP1753868A2 EP05754405A EP05754405A EP1753868A2 EP 1753868 A2 EP1753868 A2 EP 1753868A2 EP 05754405 A EP05754405 A EP 05754405A EP 05754405 A EP05754405 A EP 05754405A EP 1753868 A2 EP1753868 A2 EP 1753868A2
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EP
European Patent Office
Prior art keywords
weight
fermentation
starch
sugar
liquid medium
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Application number
EP05754405A
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German (de)
English (en)
Inventor
Markus Pompejus
Stephan Freyer
Markus Lohscheidt
Oskar Zelder
Matthias Boy
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
BASF SE
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BASF SE
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Filing date
Publication date
Application filed by BASF SE filed Critical BASF SE
Publication of EP1753868A2 publication Critical patent/EP1753868A2/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P25/00—Preparation of compounds containing alloxazine or isoalloxazine nucleus, e.g. riboflavin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/02—Amides, e.g. chloramphenicol or polyamides; Imides or polyimides; Urethanes, i.e. compounds comprising N-C=O structural element or polyurethanes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/08—Lysine; Diaminopimelic acid; Threonine; Valine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/10—Citrulline; Arginine; Ornithine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/12—Methionine; Cysteine; Cystine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/14—Glutamic acid; Glutamine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/04—Alpha- or beta- amino acids
    • C12P13/24—Proline; Hydroxyproline; Histidine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/02—Monosaccharides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/20—Preparation of compounds containing saccharide radicals produced by the action of an exo-1,4 alpha-glucosidase, e.g. dextrose
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/44—Polycarboxylic acids
    • C12P7/46—Dicarboxylic acids having four or less carbon atoms, e.g. fumaric acid, maleic acid
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/582—Recycling of unreacted starting or intermediate materials

Definitions

  • the present invention relates to the fermentative production of fine chemicals by grinding, liquefying and saccharifying starch sources and the use of the sugar solution thereby obtained as a fermentation medium.
  • Fermentative processes for the production of fine chemicals such as Amino acids, vitamins and carotenoids by microorganisms are generally known. Depending on the different process conditions, different carbon sources are used. These range from pure sucrose to beet and sugar cane molasses, so-called "high test molasses” (inverted sugar cane molasses) to glucose from starch hydrolysates. For the biotechnological production of L-lysine, acetic acid and ethanol are also mentioned as large-scale co-substrates ( Pfefferle et al., Biotechnological Manufacture of Lysine, Advances in Biochemical Engineering / Biotechnology, Vol. 79 (2003), 59-112).
  • starch An important source of carbon for the fermentative production of fine chemicals mediated by microorganisms is starch. This must first be liquefied and saccharified in upstream reaction steps before it can be used as a carbon source in a fermentation.
  • a natural starch source such as potatoes, cassava, cereals, e.g. Wheat, corn, barley, rye, triticale or rice are usually obtained in a pre-cleaned form and then liquefied and saccharified enzymatically, in order to be used in the actual fermentation for the production of fine chemicals.
  • non-pretreated starch sources for the production of carbon sources for the fermentative production of fine chemicals.
  • the starch sources are first crushed by grinding.
  • the regrind is then subjected to liquefaction and saccharification. Since this regrind naturally contains, in addition to starch, a number of non-starch-containing constituents which adversely affect the fermentation, these constituents are usually separated off before the fermentation.
  • the removal can be done either directly after grinding (WO 02/277252; JP 2001-072701; JP 56-169594; CN 1218111), after liquefaction (WO 02/277252; CN 1173541) or after saccharification (CN 1266102; Beukema et al .: Production of fermentation syrups by enzymatic hydrolysis of potatoes; potatoe saccharification to give eulture medium (Conference Abstract), Symp. Biotechnol. Res. Neth. (1983), 6; NL8302229). In all variants, however, a largely pure starch hydrolyzate is used in the fermentation.
  • Newer techniques are concerned in particular with improved methods which require a purification e.g. of liquefied and saccharified starch solutions (JP 57159500) and fermentation media from renewable resources (EP 1205557).
  • Unprocessed starch sources are known to be widely used in the fermentative production of bioethanol.
  • the process of dry grinding, liquefaction and saccharification of starch sources known as “dry milling”, is technically established on a large scale.
  • Corresponding process descriptions can be found, for example, in “The Alcohol Textbook - A reference for the beverage, fuel and industrial alcohol industries ", Jaques et al. (Ed.), Nottingham Univ. Press 1995, ISBN 1 -8977676-735, and in McAloon et al., “Determining the cost of producing ethanol from com starch and lignocellulosic feedstocks", NREL / TP-580-28893, National Renewable Energy Laboratory, October 2000.
  • whole grains are ground, preferably maize, wheat, barley, millet and rye.
  • wet milling no additional liquid is added.
  • the grinding into fine components serves to make the starch contained in the grains accessible to the action of water and enzymes in the subsequent liquefaction and saccharification.
  • the oxygen supply to the microorganisms used is a limiting factor. Little is known about the influence of high solid concentrations on the oxygen transition from the gas to the liquid phase and thus on the oxygen transfer rate. On the other hand, it is known that an increasing viscosity with increasing solids concentration leads to a reduced oxygen transfer rate. If surface-active substances are also introduced into the fermentation medium with the solids, they influence the tendency of the gas bubbles to coalesce. The resulting bubble size in turn has a significant influence on oxygen transfer (Mersmann, A. et al .: Selection and Design of Aerobic Bioreactors, Chem. Eng. Technol. 13 (1990), 357-370).
  • JP 2001/275693 describes a process for the fermentative production of amino acids, in which peeled cassava tubers are used as the starch source and have been dry milled. To carry out the process, however, it is necessary to set a particle size of the ground material of ⁇ 150 ⁇ m. In the filtration used for this purpose, more than 10% by weight of the regrind used, including components not containing starch, are separated off prior to the liquefaction / saccharification of the starch contained and the subsequent fermentation. In addition, the problem of separating non-starchy components does not arise in that the fermentation products, e.g. Lysine, are intended as a feed additive and therefore the non-starchy components of the cassava can remain in the product of value.
  • the fermentation products e.g. Lysine
  • cassava should be relatively unproblematic compared to other starch sources for dry milling. While the starch content of the cassava root in the dry state is typically at least 80% by weight (Menezes et al., Fungal celluloses as an aid for the saccharification of Cassava, Biotechnology and Bioengineering, Vol.
  • the dry starch content is significantly lower in comparison with cereals, generally below 70% by weight, for example about 68% by weight for corn and about 65 for wheat % By weight (Jaques et al., The Alcohol Textbook, see above). Accordingly, the glucose solution obtained after liquefaction and saccharification contains fewer foreign components and, in particular, fewer solids when using dry-ground cassava than when using another dry-ground starch source.
  • the viscosity of the reaction mixture is increased by an increased amount of foreign constituents.
  • starch from cassava should be relatively easy to process. Compared to maize starch, it has a higher viscosity at the swelling temperature, but the viscosity decreases more quickly with cassava than with maize starch (Menezes, TJB de, Saccharification of Cassava for ethyl alcohol production, Process Biochemistry, 1978, page 24, right column).
  • the swelling and gelatinization temperatures of starch from cassava are lower than that of starch from cereals such as maize, which is why it is more accessible to bacterial amylase than cereal starch (Menezes, T.J.B. de, op. Cit.).
  • cassava over other starch sources are its low cellulose content and its low phytate content.
  • Cellulose and hemicellulose can be converted into furfurals, especially under acidic saccharification conditions (Jaques et al., The Alcohol Textbook, see above; Menezes, T.J.B. de, see above), which in turn can have an inhibitory effect on the microorganisms used in the fermentation.
  • Phytate also inhibits the microorganisms used for fermentation.
  • cassava as a starch source in a process corresponding to dry milling
  • a process based on cassava is nevertheless complex, not optimized and therefore not widely used.
  • the invention thus relates to a process for producing at least one microbial metabolite with at least 3 C atoms or with at least 2 C atoms and at least 1 N atom by sugar-based microbial fermentation, comprising:
  • a microorganism strain producing the desired metabolic product (s) is cultivated with the sugar-containing liquid medium, which is obtained by:
  • dry corn fruits or seeds come into consideration as starch sources, which in the dried state have at least 40% by weight and preferably at least 50% by weight of starch.
  • starch sources which in the dried state have at least 40% by weight and preferably at least 50% by weight of starch.
  • the starch source is preferably selected from cereal grains, particularly preferably from maize, rye, triticale and wheat grains.
  • the method according to the invention can also be carried out with other starch sources, such as, for example, potatoes, cassava / tapioca or a mixture of different starch-containing fruits or seeds.
  • the sugars contained in the sugar-containing liquid medium are preferably monosaccharides such as hexoses and pentoses, for example glucose, fructose, mannose, galactose, sorbose, xylose, arabinose and ribose, in particular glucose.
  • monosaccharides such as hexoses and pentoses
  • glucose fructose, mannose, galactose, sorbose, xylose, arabinose and ribose
  • the proportion of monosaccharides other than glucose can depend on the starch source used and the non-starchy content contained therein. Components vary and are influenced by the process control, for example by disintegrating cellulose components by adding cellulases.
  • the monosaccharides of the sugar-containing liquid medium advantageously comprise a proportion of glucose of at least 60% by weight, preferably at least 70% by weight and particularly preferably at least 80% by weight, based on the total amount of sugar contained in the sugar-containing liquid medium.
  • the amount of glucose is usually in the range from 75 to 99% by weight, in particular from 80 to 97% by weight and especially from 85 to 95% by weight, based on the total amount of sugar contained in the sugar-containing liquid medium.
  • the sugar-containing liquid medium with which the microorganism strain producing the desired metabolic products is cultivated contains at least part, preferably at least 20% by weight, in particular at least 50% by weight, especially at least 90% by weight and very particularly at least 99% by weight .-% of the non-starchy solid components contained in the ground cereal grains, according to the degree of grinding.
  • the proportion of the non-starch-containing solid constituents in the sugar-containing liquid medium is preferably at least 10% by weight and in particular at least 25% by weight, e.g. between 25 and 75% by weight and especially between 30 and 60% by weight.
  • the respective starch source with or without the addition of liquid, e.g. Water, milled, preferably without adding liquid.
  • Dry grinding can also be combined with a subsequent wet grinding.
  • hammer mills, rotor mills or roller-grist mills are typically used; Mixers, agitator ball mills, circulation mills, disk mills, ring chamber mills, vibratory mills or planetary mills are suitable for wet grinding. Basically, other mills can also be used.
  • the person skilled in the art can determine the amount of liquid required for wet grinding in routine experiments. It is usually set so that the dry matter content is in the range from 10 to 20% by weight.
  • a suitable grain size for the subsequent process steps is set by the grinding. It has proven to be advantageous here if the ground material obtained in grinding, in particular in the case of dry grinding, in step a1) has flour particles, ie particulate constituents, with a grain size in the range from 100 to 630 ⁇ m in a proportion of 30 to 100% by weight. %, preferably 40 to 95% by weight and particularly preferably 50 to 90% by weight.
  • the ground material obtained preferably contains 50% by weight of flour particles with a grain size of more than 100 ⁇ m. As a rule, at least 95% by weight of the ground flour particles have a grain size of less than 2 mm.
  • the grain size is measured by sieve analysis using a vibration analysis machine.
  • a minor one Grain size is generally advantageous for achieving a high product yield.
  • a too small particle size can, however, lead to problems, in particular due to lump formation / agglomeration, when mashing the ground material during liquefaction or when working up, for example when drying the solids after the fermentation step.
  • Flours are usually characterized by the degree of grinding or by the type of flour, and these correlate with one another in such a way that the number of types of flour increases with the degree of grinding.
  • the degree of grinding corresponds to the amount by weight of the flour obtained, based on 100 parts by weight of the ground material used. While at first pure, finest flour, e.g. accumulates from the inside of the grain, increases the proportion of crude fiber and husk content in the flour during further grinding, i.e. with increasing degree of grinding, the starch content is reduced.
  • the degree of grinding is therefore also reflected in the so-called flour type, which is used as a figure to classify flours, in particular cereal flours, and which is based on the ash content of the flour (so-called ash scale).
  • the flour type or number of types indicates the amount of ash (minerals) in mg that remains after burning 100 g of flour dry substance.
  • a higher type number means a higher degree of grinding, since the core of the cereal grain contains about 0.4% by weight, while the shell contains about 5% by weight of ash.
  • the cereal flours mainly consist of the crushed flour body, i.e. the starch component of the cereal grains; with a higher degree of grinding, the cereal flours also contain the chopped, protein-containing aleurone layer of the cereal grains, with grist also the components of the protein and fat-containing seedling as well as the crude fiber and ash-containing seed shells.
  • Flours with a high degree of grinding or a high number of types are generally preferred for the purposes of the invention. If grain is used as a starch source, the whole unpeeled grains are preferably ground and processed further.
  • the starch source will be comminuted to a size suitable for milling before milling, for example when using larger fruits such as potatoes or cassava. In the case of grain, this crushing step can be omitted and the whole grain is used and ground.
  • At least a portion of the regrind is preferably given in step a2), preferably at least 40% by weight, in particular at least 50% by weight and very particularly preferably at least 55% by weight in the course of the liquefaction the saccharification in the reactor. Frequently, the amount added will not exceed 90% by weight, in particular 85% by weight and particularly preferably 80% by weight.
  • the partial amount of regrind added in the course of the process is preferably fed to the reactor under conditions such as are present during the liquefaction.
  • the addition can be discontinuous, ie in portions in several portions, preferably not more than 20% by weight, particularly preferably not make up more than 10% by weight, for example 1 to 20% by weight, in particular 2 to 10% by weight, of the total amount of the ground material to be liquefied, or take place continuously. It is essential to the invention that at the beginning of the liquefaction, only a part of the millbase, preferably not more than 60% by weight, in particular not more than 50% by weight and particularly preferably not more than 45% by weight of the millbase, is present in the reactor is located and the remaining amount of the ground material is added during the liquefaction.
  • the liquefaction can also be carried out continuously, for example in a multi-stage reaction cascade.
  • the liquefaction in step a2) takes place in the presence of at least one starch liquefying enzyme, which is preferably selected from ⁇ -amylases.
  • starch liquefying enzyme which is preferably selected from ⁇ -amylases.
  • Other enzymes which liquefy and stabilize starch under the reaction conditions can also be used.
  • the ⁇ -amylase (or the starch liquefying enzyme used) can be placed in the reaction vessel or added in the course of step a2).
  • a partial amount of the ⁇ -amylase required in step a2) is preferably added at the beginning of step a2) or this partial amount is placed in the reactor.
  • the total amount of ⁇ -amylase is usually in the range from 0.002 to 3.0% by weight, preferably from 0.01 to 1.5% by weight and particularly preferably from 0.02 to 0.5% by weight, based on the total amount of starch source used.
  • the liquefaction can be carried out above or below the gelation temperature.
  • the liquefaction in step a2) is preferably carried out at least temporarily above the gelation temperature of the starch used (so-called cooking process).
  • a temperature in the range between 70 and 165 ° C., preferably between 80 and 125 ° C. and particularly preferably between 85 and 115 ° C. is chosen, the temperature preferably being at least 5 ° C. and particularly preferably at least 10 ° C. above the gelation temperature.
  • step a2) is preferably carried out at least at times at a pH in the weakly acidic range, preferably between 4.0 and 7.0, particularly preferably between 5.0 and 6.5, usually before or at the beginning of step a2) the pH is adjusted; this pH value is preferably checked during the liquefaction and adjusted if necessary.
  • the pH is preferably adjusted with dilute mineral acids such as H 2 SO 4 or H 3 PO 4 or with dilute alkali solutions such as NaOH or KOH.
  • step a2) of the process according to the invention is carried out in such a way that initially a partial amount of at most 60% by weight, preferably at most 50% by weight and particularly preferably at most 45% by weight, for example 10 to 60% by weight. %, in particular 15 to 50% by weight and particularly preferably 20 to 45% by weight, based on the total amount of the ground material, in an aqueous liquid, for example fresh water, recycled process water, for example from fermentation or processing, or suspended in a mixture of these liquids and then the liquefaction is carried out.
  • an aqueous liquid for example fresh water, recycled process water, for example from fermentation or processing, or suspended in a mixture of these liquids and then the liquefaction is carried out.
  • the liquid used to produce the suspension it is possible to bring the liquid used to produce the suspension to a slightly elevated temperature, e.g. in the range of 40 to 60 ° C, to pre-temper. However, it is preferred to use the liquids at room temperature.
  • the at least one starch-liquefying enzyme preferably an ⁇ -amylase
  • ⁇ -amylase it is advantageous to add only a subset of the ⁇ -amylase, e.g. 10 to 70% by weight and in particular 20 to 65% by weight, based on the total of ⁇ -amylase used in step a2).
  • the amount of ⁇ -amylase added at this time depends on the activity of the respective ⁇ -amylase in relation to the starch source used under the reaction conditions and is usually in the range from 0.0004 to 2.0% by weight, preferably from 0.001 up to 1.0% by weight and particularly preferably from 0.02 to 0.3% by weight, based on the total amount of the starch source used.
  • the portion of the ⁇ -amylase can be mixed with the liquid used before preparing the suspension.
  • the partial amount of ⁇ -amylase is preferably heated to the temperature used for liquefaction, especially at room temperature or only a slightly elevated temperature, e.g. in the range of 20 to 30 ° C, added to the suspension.
  • the amounts of ⁇ -amylase and regrind are advantageously selected so that the viscosity is sufficiently reduced during the gelling process to enable effective mixing of the suspension, for example by means of stirring.
  • the viscosity of the reaction mixture during gelling is preferably at most 20 Pas, particularly preferably at most 10 Pas and very particularly preferably at most 5 Pas. Viscosity is usually measured using a Haake Roto Visko RV20 viscometer with M5 measuring system and MVDIN measuring device at a temperature of 50 ° C and a shear rate of 200 s "1 .
  • the suspension thus prepared is then preferably heated to a temperature above the gelation temperature of the starch used.
  • a temperature in the range between 70 and "165 ° C, preferably between 80 and 125 ° C, and more preferably selected from 85 to 115 ° C, with the temperature pre preferably at least 5 ° C, and most preferably at least 10 ° C.
  • further portions of the starch source for example 2 to 20% by weight and in particular 5 to 10% by weight, based on the total amount of starch used, are gradually added to the starch-containing suspension. It is preferred to add those in the course of liquefaction.
  • the portion of the grinding stock not used when preparing the suspension can be added continuously during the liquefaction.
  • the temperature should advantageously be kept above the gelation temperature of the starch during the addition.
  • the reaction mixture is usually left for a while, e.g. 30 to 60 minutes or longer, if necessary, kept at the temperature set above the gelation temperature of the starch, i.e. overcooked.
  • the reaction mixture is then typically brought to a slightly lower temperature above the gelation temperature, e.g. 75 to 90 ° C, cooled, before another portion of ⁇ -amylase, preferably the majority, is added.
  • the amount of ⁇ -amylase added at this time is preferably 0.002 to 2.0% by weight, particularly preferably from 0.01 to 1.0% by weight and very particularly preferably from 0.02 to 0.4% by weight, based on the total amount of the starch source used.
  • the reaction mixture is kept at the set temperature or, if appropriate, heated further until the starch detection with iodine or possibly another test for the detection of starch is negative or at least substantially negative. If necessary, one or more further subsets of ⁇ -amylase, e.g. in the range of 0.001 to 0.5% by weight and preferably 0.002 to 0.2% by weight, based on the total amount of the starch source used, are added to the reaction mixture.
  • saccharification of the dextrins contained in the liquid medium is carried out continuously or discontinuously, preferably continuously.
  • the liquefied medium can be completely saccharified in a special saccharification tank before it is fed to the fermentation step b).
  • it has proven advantageous to carry out only partial saccharification before fermentation For example, one can proceed in such a way that a subset of the dextrins contained in the liquid medium, for example in the range from 10 to 90% by weight and in particular in the range from 20 to 80% by weight, based on the total weight of the dextrins ( or the original starch), saccharified and the resulting sugar-containing liquid medium is used in the fermentation.
  • saccharification can then take place in situ in the fermentation medium.
  • the saccharification can also be carried out directly in the fermenter (in situ) without the need for a separate saccharification tank.
  • the advantages of in-situ saccharification, ie saccharification partially or completely in the fermenter, are on the one hand reduced investment costs, and on the other hand a delayed release of the glucose can possibly result in a higher glucose concentration in the batch (batch) without any inhibition or Metabolism change of the microorganisms used occurs.
  • too high a concentration of glucose for example, leads to the formation of organic acids (acetate), while Saccharomyces cerevisae in this case switches to fermentation, for example, although there is sufficient oxygen in aerated fermenters (crabtree effect).
  • a delayed release of glucose can be adjusted by regulating the glucoamylase concentration. As a result, the aforementioned effects can be suppressed and more substrate can be placed in front, so that the dilution resulting from the feed stream supplied can be reduced
  • the liquefied starch solution is usually brought up to or slightly below the temperature optimum of the saccharifying enzyme, e.g. cooled to 50 to 70 ° C, preferably 60 to 65 ° C or tempered and then mixed with glucoamylase.
  • the liquefied starch solution is usually brought up to the fermentation temperature, ie. H. 32 to 37 ° C, cool before feeding them to the fermenter.
  • the glucoamylase (or the at least one saccharifying enzyme) for saccharification is added directly to the fermentation broth.
  • the saccharification of the liquefied starch according to step a2) takes place parallel to the metabolism of the sugar by the microorganisms according to step b).
  • the pH of the liquid medium is advantageously set to a value in the optimal range of action of the glucoamylase used, preferably in the range between 3.5 and 6.0; particularly preferably between 4.0 and 5.5 and very particularly preferably between 4.0 and 5.0.
  • panthothenate and vitamin B 2 for example, this can be advantageous overall despite the restricted activity of standard glucoamylases in this pH range or be necessary due to the fermentation conditions to be set.
  • saccharification takes place in a special saccharification tank.
  • the liquefied starch solution is heated to a temperature which is optimal for the enzyme or slightly below it, and the pH is adjusted to a value which is optimal for the enzyme in the manner described above.
  • the glucoamylase is usually added to the liquid medium containing dextrin in an amount of from 0.001 to 5.0% by weight, preferably from 0.005 to 3.0% by weight and particularly preferably from 0.01 to 1.0% by weight, based on the total amount of starch source used.
  • the dextrin-containing suspension is preferably kept at the set temperature for a period of, for example, 2 to 72 hours or longer, if necessary in particular from 5 to 48 hours, the dextrins being saccharified to monosaccharides.
  • the progress of saccharification can be followed using methods known to those skilled in the art, for example HPLC, enzyme tests or glucose test sticks. The saccharification is complete when the concentration of the monosaccharides no longer rises or falls significantly.
  • the discontinuous or continuous, preferably the discontinuous and in particular portionwise addition of the millbase takes place in the presence of the at least one ⁇ -amylase and the at least one glucoamylase in step a2) in such a way that the viscosity of the liquid medium is at most 20 Pas, preferably is a maximum of 10 Pas and particularly preferably a maximum of 5 Pas.
  • the viscosity control it has proven to be advantageous if at least 25% by weight, preferably at least 35% by weight and particularly preferably at least 50% by weight of the total amount of the millbase added at a temperature above the gelatinization temperature of the millbase Starch can be added.
  • the control of the viscosity can further be influenced by adding the at least one starch liquefying enzyme, preferably an ⁇ -amylase, and / or the at least one saccharifying enzyme, preferably a glucoamylase, itself in portions.
  • the sugar-containing liquid With a monosaccharide content of preferably more than 30% by weight, particularly preferably more than 35% by weight and very particularly preferably more than 40% by weight ,
  • all ⁇ -amylases can be used to liquefy the starch content in the regrind, in particular ⁇ -amylases obtained from Bacillus lichenformis or Bacillus staerothermophilus and especially those that liquefy by dry milling processes obtained materials can be used in the production of bioethanol.
  • the ⁇ -amylases suitable for liquefaction are also commercially available, for example from Novozymes under the name Termamyl 120 L, type L; or from Genencor under the name Spezyme.
  • a combination of different ⁇ -amylases can also be used for liquefaction.
  • all glucoamylases can be used to saccharify the dextrins (ie oligosaccharides) in the liquefied starch solution.
  • the, in particular glucoamylases which were obtained from Aspergilus and especially those which are used for saccharification of materials obtained by dry-milling processes in the context of the production of bioethanol.
  • the glucoamylases suitable for saccharification are also commercially available, for example from Novozymes under the name Dextrozyme GA; or from Genencor under the name Optidex.
  • a combination of different glucoamylases can also be used.
  • the concentration of Ca 2+ ions can be adjusted to an enzyme-specific optimal value, for example with CaCl 2 . Suitable concentration values can be determined by the person skilled in the art in routine experiments. If, for example, termamyl is used as the ⁇ -amylase, it is advantageous to set a Ca 2+ concentration in the liquid medium of, for example, 50 to 100 ppm, preferably 60 to 80 ppm and particularly preferably about 70 ppm.
  • the whole starch source e.g. in the case of grain the whole grain is ground, this also includes the non-starchy solid components of the starch source. This often requires the entry of a not negligible proportion of phytate from the corn crop.
  • at least one phytase is advantageously added to the liquid medium in step a2) before the sugar-containing liquid medium is fed to the fermentation step b).
  • the phytase can be added before, during or after the liquefaction or the sugarification, provided that it has the required heat stability.
  • Phytases can be used, provided their activity is at most insignificantly restricted under the reaction conditions.
  • the amount of phytase is usually 1 to 10,000 units / kg of starch source and in particular 10 to 2000 units / kg of starch source.
  • enzymes for example pullulanases, cellulases, hemicellulases, glucanases, xylanases, glucosidases or proteases, can also be added to the reaction mixture during the production of the sugar-containing liquid medium.
  • the addition of these enzymes can have a positive influence on the viscosity, ie reduce it (for example by cleavage of longer-chain glucans and / or (arabino-) xylans), the release of metabolizable glucosides and the release of (residual) starch.
  • the use of proteases has analogous positive effects, whereby additional amino acids can be released as growth factors for the fermentation.
  • the sugar-containing liquid medium can advantageously be used for the fermentative production of a microbial metabolite with at least 3 C atoms or with at least 2 C atoms and at least 1 N atom.
  • the sugar-containing liquid medium produced in step a) is fed to a fermentation according to b).
  • fine chemicals, ie compounds with at least 3 carbon atoms and / or at least one nitrogen atom and at least 2 carbon atoms are produced by the microorganisms.
  • the fermentation process can generally be carried out in the customary manner known to the person skilled in the art.
  • the volume ratio of the supplied sugar-containing liquid medium to the liquid medium which is present and which contains the microorganisms is generally in the range from about 1:10 to 10: 1, for example about 1: 2 or about 2: 1 and in particular about 1: 1.
  • the sugar content in the fermentation broth can be regulated via the feed rate of the sugar-containing liquid medium.
  • the feed rate will be adjusted so that the monosaccharide content in the fermentation broth is in the range from 0 0% by weight to about 5% by weight; however, the fermentation can also be carried out at significantly higher monosaccharide contents in the fermentation broth, for example about 10 to 20% by weight.
  • the sugar-containing liquid medium obtained in step a) can optionally be sterilized before the fermentation, the microorganisms being killed by thermal, chemical or mechanical methods.
  • the broth is usually heated to temperatures above 80 ° C.
  • the cells can be killed or lysed immediately before the fermentation.
  • the entire sugar-containing liquid medium is fed to the lysis or killing. This can be done thermally, mechanically or chemically.
  • a preferred embodiment of the invention relates to a method in which the liquid medium obtained in step a) is immediate, i.e. without prior sterilization, the fermentation is carried out or an at least partial in-situ saccharification is carried out.
  • the fermentation results in a liquid medium which, in addition to the desired non-volatile microbial metabolite, essentially the biomass produced during the fermentation, the non-metabolized components of the saccharified starch solution and in particular the non-starch-containing solid components of the starch source, such as fibers and unused sugar , and contains unused buffer and nutrient salts.
  • This liquid medium is also referred to in the present application as a fermentation broth, the expression fermentation broth also encompassing the (sugar-containing) liquid medium in which there is only a partial or incomplete fermentative conversion of the sugars contained therein, ie a partial or incomplete microbial metabolism of the monosaccharides ,
  • fine chemical in the following includes in particular organic, optionally 1 or more, e.g.
  • B 1, 2, 3 or 4 hydroxyl-bearing mono-, di- and tricarboxylic acids with preferably 3 to 10 carbon atoms, for example tartaric acid, itaconic acid, succinic acid, fumaric acid, maleic acid, 2,5-furanedicarboxylic acid, 3-hydroxypropionic acid, glutaric acid, Levulinic acid, lactic acid, propionic acid, gluconic acid, aconitic acid and diaminopimelic acid, citric acid; proteinogenic and non-proteinogenic amino acids, for example lysine, glutamate, methionine, phenylalanine, aspartic acid and threonine; Purine and pyrimidine bases; Nucleosides and nucleotides, for example nicotinamide adenine dinucleotide (NAD) and adenosine 5'-monophosphate (AMP); lipids; saturated and unsaturated fatty acids with preferably 10 to 22 carbon atom
  • cofactor includes non-proteinaceous compounds that are necessary for normal enzyme activity to occur. These compounds can be organic or inorganic; the cofactor molecules according to the invention are preferably organic. Examples of such molecules are NAD and nicotinamide adenine dinucleotide phosphate (NADP); the precursor of these cofactors is niacin.
  • the term "nutraceutical” encompasses food additives which are beneficial to plants and animals, in particular humans. Examples of such molecules are vitamins, antioxidants and certain lipids, e.g. Polyunsaturated fatty acids.
  • the metabolic products produced include 3 to 10 enzymes, amino acids, vitamins, disaccharides, aliphatic mono- and dicarboxylic acids C atoms, aliphatic hydroxycarboxylic acids with 3 to 10 C atoms, ketones with 3 to 10 C atoms, alkanols with 4 to 10 C atoms, alkane diols with 3 to 8 C atoms and polyhydroxyalkanoates.
  • non-volatile metabolic products are understood to mean compounds which, in general, cannot be removed from the fermentation broth without being decomposed by distillation. These compounds generally have a boiling point above the boiling point of water, often above 150 ° C and in particular above 200 ° C at normal pressure. As a rule, these are compounds which are present in a solid state under normal conditions (298 K, 101, 3 kPa).
  • non-volatile microbial metabolites which have a melting point below the boiling point of water or / and an oily consistency at normal pressure.
  • the maximum temperature will generally be checked during processing, especially during drying.
  • These compounds can also advantageously be produced by formulating them in quasi-solid form (pseudo-solid form) on adsorbents. As a rule, the solid constituents of the fermentation broth are separated off in this case before the depletion or isolation of the valuable product in step c).
  • Suitable adsorbents for the aforementioned purpose are, for example, activated carbons, aluminum oxides, silica gels, silica, clay, carbon blacks, zeolites, inorganic alkali and alkaline earth metal salts such as sodium, potassium, magnesium and calcium hydroxides, carbonates, silicates, sulfates and phosphates , in particular magnesium and calcium salts, for example Mg (OH) 2 , MgCO 3 , MgSiO, CaSO 4 , CaCO 3) alkaline earth metal oxides, for example MgO and CaO, other inorganic phosphates and sulfates, for example ZnSO, salts of organic acids, in particular their alkali and Alkaline earth metal salts and especially their sodium and potassium salts, for example sodium and potassium acetate, formate, hydrogen formate and citrate, as well as higher molecular weight organic carriers such as carbohydrates, for example sugar, optionally modified starches, cellulose, lignin, and generally
  • the carrier materials mentioned will have no or only small proportions, in particular only traces of halogens such as chloride ions and of nitrates.
  • examples of compounds which have a melting point below the boiling point of water or / and an oily consistency at normal pressure and which can advantageously be prepared in this way by the process according to the invention are ⁇ -linolenic acid, dihomo- ⁇ -linolenic acid, arachidonic acid, eicosapentaenoic acid and Docosahexaenoic acid, also propionic acid, lactic acid, propanediol, butanol and acetone.
  • these compounds in pseudo-solid formulation are also understood to be non-volatile microbial metabolites in solid form.
  • microorganisms used in the fermentation depend in a manner known per se on the respective fine chemicals, as explained in detail below. They can be of natural origin or genetically modified. Examples of suitable microorganisms and fermentation processes are given in Table A below:
  • Preferred embodiments of the method according to the invention relate to the production of enzymes such as phytases, xylanases, glucanases; Amino acids such as lysine, methionine, threonine; Vitamins such as pantothenic acid and riboflavin; Precursors and follow-up products thereof; and the preparation of the abovementioned mono-, di- and tricarboxylic acids, in particular aliphatic mono- and dicarboxylic acids with 3 to 10 carbon atoms such as propionic acid and succinic acid, aliphatic hydroxycarboxylic acids with 3 to 10 carbon atoms such as lactic acid; of the aforementioned longer-chain alkanols, in particular alkanols having 4 to 10 carbon atoms, such as butanol; of the aforementioned diols, in particular alkane diols with 3 to 8 carbon atoms, such as propanediol; of the aforementioned ketones, in
  • the microorganisms used in the fermentation are therefore selected from natural or recombinant microorganisms which produce at least one of the following metabolic products: enzymes such as phytase, xylanase, glucanase; Amino acids such as lysine, threonine and methionine; Vitamins such as pantothenic acid and riboflavin; Precursors and / or derived products thereof; Disaccharides such as trehalose; aliphatic mono- and dicarboxylic acids with 3 to 10 carbon atoms such as propionic acid and succinic acid; aliphatic hydroxycarboxylic acids with 3 to 10 carbon atoms such as lactic acid; Ketones with 3 to 10 carbon atoms such as acetone; Alkanols with 4 to 10 carbon atoms, such as butanol; Alkanediols with 3 to 8 carbon atoms, such as propanediol; and polyhydroxy
  • the microorganisms are selected from the genera Corynebacterium, Bacillus, Ashbya, Escherichia, Aspergillus, Alcaligenes, Actinobacillus, Anaerobiospirillum, Lactobacillus, Propionibacterium and Clostridium, in particular re among strains of Corynebacterium glutamicum, Bacillus subtilis, Ashbya gossypii, Escherichia coli, Aspergillus niger, or Alcaligenes latus, Anaerobiospirillum succiniproducens, Actinobacillus succinogenes, Lactobacillus delbschreibii, Lactobacillus leichmannii, Propionibacterium arabinosum, Propionibacterium schermanii, Propionibacterium freudenreichii, Clostridium propionicum and Clostridium acetobutlicum ,
  • the metabolic product produced by the microorganisms in the fermentation is lysine.
  • Analogous conditions and procedures as described for other carbon sources can be used to carry out the fermentation, e.g. in Pfefferle et al., loc. cit. and US 3,708,395.
  • both a continuous and a discontinuous (batch or fed-batch) mode of operation come into consideration; the fed-batch mode of operation is preferred.
  • the metabolic product produced by the microorganisms in the fermentation is methionine.
  • Analogous conditions and procedures as described for other carbon sources can be used to carry out the fermentation, e.g. in WO 03/087386 and WO 03/100072.
  • the metabolic product produced by the microorganisms in the fermentation is pantothenic acid.
  • Analogous conditions and procedures as described for other carbon sources can be used to carry out the fermentation, e.g. in WO 01/021772.
  • the metabolic product produced by the microorganisms in the fermentation is polyhydroxyalkanoates such as poly-3-hydroxybutyrate and copolyesters with other organic hydroxycarboxylic acids such as 3-hydroxyvaleric acid, 4-hydroxybutyric acid and others which are used in Steinbüchel (loc. Cit.) Are described, e.g. also longer-chain hydroxycarboxylic acids such as 3-hydroxyoctanoic acid, 3-hydroxydecanoic acid and 3-hydroxytetradecanoic acid, and mixtures thereof. Analogous conditions and procedures as described for other carbon sources can be used to carry out the fermentation, e.g. in S. Y. Lee, Plastic Bacteria? Progress and prospects for polyhydroxyalkanoate production in bacteria, Tibtech, Vol. 14, (1996), pp. 431-438.
  • the metabolic product produced by the microorganisms in the fermentation is riboflavin.
  • Analogous conditions and procedures as described for other carbon sources can be used to carry out the fermentation, for example in WO 01/011052, DE 19840709, WO 98/29539, EP 1186664 and Fujioka, K .: New biotechnology for riboflavin (vitamin B2) and character of this ri- boflavin. Fragrance Journal (2003), 31 (3), 44-48.
  • the metabolic product produced by the microorganisms in the fermentation is a phytase.
  • Analogous conditions and procedures as described for other carbon sources can be used to carry out the fermentation, e.g. in WO 98/55599.
  • a sterilization step is optionally carried out in the manner described above.
  • the isolation or depletion of the metabolic product from the fermentation broth according to step c) is generally carried out in such a way that at least one metabolic product is depleted or isolated from the fermentation broth in such a way that the content of this metabolic product in the remaining fermentation broth is at most 20% by weight, in particular at most 10% by weight, especially at most 5% by weight and very particularly at most 2.5% by weight, in each case based on the total weight of the remaining fermentation broth.
  • the isolation or depletion of fine chemicals (i.e. the microbial metabolite) from the fermentation broth according to step c) can be carried out in one or more steps.
  • An important step in this is the separation of the solid components from the fermentation broth. This can be done either before or after the isolation of the valuable product.
  • Solid-liquid phase separation conventional methods are known in the art, which also include steps for the coarse and fine cleaning of the valuable materials as well as for packaging (e.g. described in Belter, P. A, Bioseparations: Downstream Processing for Biotechnology, John Wiley & Sons (1988 ), and Ullmann's Encyclopedia of Industrial Chemistry, 5th edition on CD-ROM, Willey-VCH).
  • the product of value can advantageously proceed by first removing the solid constituents from the fermentation broth, for example by means of centrifugation or filtration, and then isolating the product of value from the liquid phase, for example by crystallization, precipitation, adsorption or distillation.
  • the product of value can also be isolated directly from the fermentation broth, for example by using chromatographic processes or extraction processes. Ionic exchange chromatography, in which the product of value can be selectively isolated on the chromatography column, should be mentioned in particular as a chromatographic method.
  • the solids are advantageously separated off from the remaining fermentation broth, for example by decanting, evaporating or drying.
  • Usual filtration methods are e.g. cake and depth filtration (e.g.
  • centrifugation methods are e.g. in G. Hultsch, H. Wilkesmann, "Filtering Centrifuges,” in D.B. Purchas, Solid - Liquid Separation, Upland Press, Croydon 1977, pp. 493-559; and H. Trawinski, The Equivalent Clarification Area of Centrifuges, Chem. Ztg. 83 (1959) 606-612.
  • Various designs, such as tube and basket centrifuges, as well as special push and inverted filter centrifuges and plate separators can be used.
  • Usual extraction methods include batch-wise or step-wise and differential continuous methods in cocurrent or countercurrent. You can work with two or one moving phases.
  • the solubility of the valuable substances and the secondary components to be separated in both phases can be influenced, among other things, by the choice of solvent, by variation of the counterions and by variation of the pH (Treybal, RE, Mass Transfer Operations, 3rd edition, New York, Mc Graw-Hill, 1979; Kula, M., Kroner, KH, Hustedt, H. and Schutee, H., Technical aspects of extractive enzyme purification, Ann. NY Acad. Sei., 341 (1981); Robinson, RG, and Cha, DY, Controlled pH extraction in the Separation of weak acids and bases, Biotech. Progress, 1 (1), 18 (1985)).
  • activated carbons In addition to many other adsorbents, activated carbons, ion exchange resins, natural or synthetic zeolites and activated aluminum oxides can be used. In addition, methods for affinity adsorption can also be used (e.g. described in Arnold, F.H., Blanch, H.W., and Wilke, C.R., Analysis of Affinity separations. Chem. Engr. J., 30, B9 (1985)).
  • Chromatography precipitation, ultra-, micro- and nanofiltration, reverse osmosis, electrophoresis, electrodialysis and isoelectric focusing, for example, can be used in particular for cleaning the fine chemicals.
  • Chromatographic processes can be carried out batchwise or continuously.
  • Continuous chromatography includes, for example, a Continuous Rotating Annular Chromatograph (CRAC) (e.g. described in AJP Martin, Discuss. Farra day Soc. 7 (1949)), a True Moving Bed Chromatograph (TMBC) (e.g. described in K. Takeuchi, T Miyauchi, Y. Uraguchi, J. Chem. Eng.
  • CRAC Continuous Rotating Annular Chromatograph
  • TMBC True Moving Bed Chromatograph
  • the valuable substances or the secondary components can be precipitated (J. W. Mullin: Crystallization, 3rd ed., Butterworth-Heinemann, Oxford 1993).
  • the precipitation can e.g. can be initiated by adding another solvent, adding salts and varying the temperature.
  • the resulting precipitate can be separated from the broth by the usual methods for separating solids described above.
  • microporous AS Michaels: “Ultrafiltration,” in ES Perry (ed.): Progress in Separation and Purification, vol. 1, Interscience Publ., New York 1968 .
  • homogeneous J. Crank, GS Park (eds.): Diffusion in Polymers, Academic Press, New York 1968; SA Stern: “The Separation of Gases by Selective Permeation,” in P.
  • Typical materials are cellulose esters, nylon, polyvinyl chloride, acrylonitrile, polypropylene, polycarbonate and ceramics. These membranes are used as a plate module (RF Madsen, Hyperfiltration and Ultrafiltration in Plate-and-Frame Systems, Elsevier, Amsterdam 1977), spiral module (US 3 417 870, 1968 (DT Bray)), tube bundle or hollow fiber module (H Strathmann: “Synthetic Membranes and their Preparation,” in MC Porter (ed.): Handbook of Industrial Membrane Technology, Noyes Publication, Park Ridge, NJ 1990, pp. 1-60). In addition, the use of liquid membranes is possible (NN Li: “Permeation Through Liquid Surfactant Membranes," AlChE J.
  • the desired material can be enriched on the feed side and via the retentate stream discharged and depleted on the feed side and discharged via the filtrate / permeate stream.
  • Electrophoretic processes are e.g. in Rudge, SR, Ladisch, MR, Process Consolidations for scale-up of liquid chromatography and electrophoresis, in Separation Coverage and Purification in Biotechnology, J. Asenjo and J. Hong, ed., ACS Symposium Series, 314, 122 (1986).
  • Numerous variants such as Isoelectric focusing in granulated gel layers, continuous isoelectric focusing with recycling, the "Rotofor" cell, free-flow focusing with recycling and multi-compartmentalized electrolysis with isoelectric membranes are used.
  • the matrix materials include Cellulose acetate, agarose gels and polyacrylamide gels are used.
  • Crystallization can be initiated, for example, by cooling, evaporation, vacuum crystallization (adiabatic cooling), reaction crystallization or salting out. Crystallization can be carried out, for example, in stirred and unstirred vessels, in the direct-contact process, in evaporation crystallizers (RK Multer, Chem Eng.
  • drying processes include processes for convection drying, for example in a drying oven, tunnel dryer, belt dryer, disc dryer, jet dryer, fluidized-bed dryer, ventilated and rotating drum dryer, spray dryer, current dryer, cyclone dryer, mixer dryer, paste grinding dryer, grinding dryer, ring dryer, shaft dryer, rotary tube dryer Carousel dryer.
  • Other processes use contact drying, eg paddle dryer; Vacuum or freeze drying, cone dryer, suction filter dryer, disc dryer, thin-film contact dryer, drum dryer, tough phase dryer, plate dryer, spiral conveyor dryer, double-cone dryer; or heat radiation (infrared, eg infrared rotary tube dryer) or dielectric energy (microwaves) for drying.
  • the drying apparatus used for thermal drying processes are mostly heated by steam, oil, gas or electric current and, depending on the design, can be operated in part under vacuum.
  • formulation processes can also be used, as described below for the production of the protein composition. These also include the addition of formulation adjuvants as indicated below.
  • the fine chemicals are isolated from the fermentation broth in accordance with c) by means of ion-exchange chromatography.
  • the general conditions and procedures are known to the person skilled in the art and e.g. in Römpp Lexikon der Chemie, 10th edition, 1997, Georg Thieme Verlag, Stuttgart; Weis, Handbuch der lonenchromatographie, 1991, VCH Verlagsgesellschaft, Weinheim.
  • the procedure will be such that the compound produced by the microorganisms is selectively bound on the ion exchanger and the ion exchanger is washed before the elution of the compound produced by the microorganisms, e.g. with water.
  • the solids can optionally be removed using customary methods known to those skilled in the art, e.g. Filtration and centrifugation are separated.
  • the solids are not separated off before the solids-laden fermentation broth is placed on the ion-exchange chromatography column.
  • the ion exchanger is advantageously flowed against by gravity with the solids-laden fermentation broth, so that the solids contained do not lead to blocking (i.e. blockage) of the ion exchange column.
  • the metabolic product produced by the microorganisms is a basic amino acid
  • this can advantageously be separated from the fermentation broth by ion exchange chromatography, using an acidic cation exchange column.
  • the basic amino acid e.g. Lysine
  • the basic amino acid can be cleaned by washing, especially with water.
  • the basic amino acid is then eluted with a suitable eluent, for example ammonia water, preferably with 5% by volume ammonia water.
  • ion exchange chromatography for the separation or purification of basic amino acids such as lysine is e.g. in WO 01/072689 and Lee et al., The use of ion exclusion chromatography as approved to the normal ion exchange chromatography to achieve a more efficient lysine recovery from fermentation broth, Enzyme and Microbial Technology 30 (2002), 798-303 ,
  • the remaining fermentation residue can be worked up in an analogous manner as described below, i.e. are treated or processed, whereby a protein-containing by-product is obtained.
  • the isolation of the valuable product is advantageously carried out by centrifugation or filtration.
  • Analogous conditions and procedures can be used, such as those used for other carbon sources e.g. were described in the earlier application DE 10359668.2.
  • the fermentation broth produced is heated in order to dissolve all of the methionine.
  • the solids are then separated off by centrifugation or filtration.
  • the clear running of the solid separation is preferably concentrated by partial or complete evaporation, the methionine crystallizing out. Finally, the methionine is dried, if necessary after a previous further filtration step.
  • the solids separated by centrifugation or filtration essentially contain the biomass produced during the fermentation and the non-metabolized components of the saccharified starch solution, e.g. Fibers. This remaining fermentation residue can be treated or processed in an analogous manner as described below, so that a protein-containing by-product is obtained.
  • the isolation of the valuable product is advantageously also carried out by centrifugation or filtration.
  • Analogous conditions and procedures can be used, as described for other carbon sources, for example in EP 1050219 and WO 01/83799. Otherwise, the work-up can be carried out in a corresponding manner, as previously described for methionine.
  • the entire fermentation broth is preferably additionally pasteurized before the solids are separated off.
  • the clear running of the solids separation is preferably partially evaporated, optionally mixed with calcium chloride and dried, preferably spray-dried.
  • pantothenic acid one can also proceed in such a way that after step c) the cells and the undissolved or solid non-starchy constituents are separated off using decanters, centrifuges, filter technology or membrane technology (microfiltration, ultrafiltration, nanofiltration) and / or one Combination of these procedures is carried out.
  • the low-solids or low-solids stream contains pantothenic acid. This stream can, for example, be further concentrated and / or be dried or formulated.
  • the solids-containing stream can be worked up, analogously as described below, to a protein-containing by-product.
  • the cells are lysed or killed. This can be done immediately after fermentation. For this purpose, the entire fermentation broth is fed to lysis or killing. This can be done thermally, mechanically or chemically.
  • the cells can also be lysed after the solids have been separated off. In this case, only the solids-rich stream is fed to the aforementioned lysis.
  • the processing of pantothenic acid is carried out by thermally killing the cells after the fermentation and separating the cells and the non-starchy solid components by means of decanters, centrifuges, filter technology or membrane technology and / or a combination thereof ,
  • the stream which is low in solids or free of solids, contains pantothenic acid.
  • This current can e.g. be further concentrated.
  • auxiliaries such as those mentioned below are advantageously added to the low-solids broth. In this way, potential foaming and / or deposit formation can be reduced.
  • the concentrated stream is then fed directly to the drying or formulation.
  • the auxiliaries mentioned below can be added before, during or after drying or formulation. This can reduce the hygroscopicity of the product, improve the flow behavior of the product and / or increase the storage stability.
  • the solids-containing stream is preferably processed into a protein-containing by-product, analogously to that described below.
  • pantothenic acid Another preferred embodiment for working up the pantothenic acid provides that auxiliaries with special cations can also be added during the fermentation. This is e.g. described in WO 02/072857.
  • pantothenic acid from the fermentation broth by means of electrodialysis or ion exchange.
  • electrodialysis or ion exchange it is also possible to remove pantothenic acid from the fermentation broth by means of electrodialysis or ion exchange.
  • these methods are not preferred, since problems may be expected.
  • the fermentation residue remaining after isolation or depletion of the pantothenic acid, ie in particular the separated solids, can generally be treated or processed in a manner analogous to that described below, so that a protein-containing by-product is obtained.
  • the isolation of the valuable product is advantageously carried out by extraction with a solvent, such as. B described in US 4310684 or EP 355307.
  • the remaining solids can in the usual manner, for. B. by filtration or centrifugation. Otherwise, the solids can be worked up in a corresponding manner, as previously described for methionine.
  • the entire fermentation broth is preferably additionally pasteurized before the solids are separated off.
  • the clear running of the solids separation is preferably partially evaporated, calcium chloride optionally added and dried, preferably spray-dried.
  • the further purification of the polyhydroxyalkanoates is carried out in a manner known per se, e.g. in US 4310684 or EP 355307.
  • the remaining fermentation residue i.e. in particular the separated solids can be treated or processed in an analogous manner as described below, so that a protein-containing by-product is obtained.
  • the remaining fermentation broth essentially contains the biomass produced during fermentation, the non-metabolized components of the saccharified starch solution, e.g. Fibers and unused sugar, as well as unused buffer and nutrient salts.
  • these solids can be obtained from the remaining fermentation broth in a manner similar to the by-product from the production of bioethanol (which is called “Distiller's Dried Grains with Solubles (DDGS)" and is marketed as such).
  • the protein-containing by-product obtained in this way can be used as feed or feed additive for feeding animals, preferably of agricultural animals, both before and after further processing or processing steps. particularly preferably from cattle, pigs and poultry, very particularly preferably from cattle.
  • the fermentation broth can be worked up or processed into a protein composition by methods known to the person skilled in the art, in particular by changing the dry substance content (for example by drying or evaporation), grinding and formulating (for example adding additives, shaping processes such as pelleting and extruding). respectively. Furthermore, the processing and processing of the by-product also includes mixing with other feed or feed additives, for example in order to standardize the levels of nutrients.
  • the protein composition is generally produced by at least partially removing the volatile constituents of the fermentation broth following the depletion or isolation of at least one metabolic product in step c). This gives a protein composition in solid or semi-solid form.
  • the content of the depleted or isolated metabolic product in the remaining fermentation broth is generally at most 20% by weight, in particular at most 15% by weight, especially at most 10% by weight and very particularly at most 5% by weight, in each case based on the total weight of the remaining fermentation broth.
  • the entire remaining broth is usually either partially evaporated in a generally multi-stage evaporation and the solids present are then e.g. separated with a decanter or the solids are separated directly from the entire fermentation broth. Centrifugation, filtration, microfiltration, ultrafiltration, nanofiltration, reverse osmosis or a combination of these methods, e.g. in a multi-stage system.
  • the solids separated off generally have a dry matter content in the range from 10 to 80% by weight, preferably 15 to 60% by weight and particularly preferably 20 to 50% by weight.
  • the finished protein composition obtained by further processing or processing advantageously has a content of about 90% by weight dry matter, so that the risk of spoilage during storage is reduced.
  • the protein composition can also be obtained by concentrating the solid constituents of the fermentation broth remaining after step c) by means of thermal processes (for example evaporation), mechanical processes (for example using filters, decanters, centrifuges) and the combinations of the individual processes mentioned which are customary for the person skilled in the art be won.
  • thermal processes for example evaporation
  • mechanical processes for example using filters, decanters, centrifuges
  • combinations of the individual processes mentioned which are customary for the person skilled in the art be won.
  • pasty residue which still contains the metabolic product according to the invention in small amounts, usually in the range from 0 to 10% by weight and in particular in the range from 0 to 5% by weight, based on the total weight of the residue, and the non-volatile , as a rule solid non-starch components of the starch source or at least large parts thereof, often at least 90% by weight or the total amount of solid non-starch components of the starch source and the biomass from the fermentation.
  • This semi-solid or solid residue can be fed directly to drying or formulation in the same way as the fermentation broth which is not concentrated and which remains after step c).
  • the liquid phase separated when the by-product is obtained can be partially recycled as process water.
  • This recirculated part of the liquid phase can advantageously be used, for example, in whole or in part in the production of the sugar-containing liquid after step a) or for the preparation of buffer or nutrient Salt solutions for use in fermentation can be used.
  • buffer or nutrient Salt solutions for use in fermentation can be used.
  • the proportion of recycled process water when preparing the suspension for starch liquefaction is therefore preferably at most 75% by weight, preferably at most 60% by weight and particularly preferably at most 50% by weight, in each case based on the total amount used to prepare the suspension Water, limited.
  • the proportion of process water when preparing the suspension in the preferred embodiment of step a2) is in the range from 5 to 60% by weight and preferably from 10 to 50% by weight, in each case based on the total for preparing the suspension water used.
  • the portion of the liquid phase not returned to the process can be concentrated to a syrup in a multi-stage evaporation.
  • the syrup thus obtained generally has a dry matter content in the range from 20 to 90% by weight, preferably 30 to 80% by weight and particularly preferably 40 to 70% by weight.
  • This syrup can be mixed with the solids separated during decanting (or in some other way) and then dried. Drying can e.g. by means of a drum dryer, spray dryer or paddle dryer, preferably a drum dryer is used.
  • the drying is preferably carried out in such a way that the solid obtained has a residual moisture content of at most 30% by weight, preferably at most 20% by weight and particularly preferably at most 10% by weight.
  • the properties of the dried by-product i.e. the protein composition
  • the properties of the dried by-product i.e. the protein composition
  • the properties of the dried by-product can be targeted with regard to various parameters such as grain size, particle shape, tendency to dust, hygroscopicity, stability , in particular storage stability, color, smell, flow behavior, tendency to agglomeration, electrostatic charging, sensitivity to light and temperature, mechanical stability and redispersibility can be assembled in a manner known per se.
  • formulation aids include e.g. Binders, carrier materials, powdering / flow aids, also color pigments, biocides, dispersants, anti-foaming agents, viscosity regulators, acids, alkalis, antioxidants, enzyme stabilizers, enzyme inhibitors, adsorbates, fats, fatty acids, oils or mixtures thereof.
  • Formulation aids of this type are advantageously used as drying aids, in particular when using formulation and drying processes such as spray, fluidized bed and freeze drying.
  • binders are carbohydrates, in particular sugars such as mono-, di-, oligo- and polysaccharides, for example dextrins, trehalose, glucose, glucose syrup, maltose, sucrose, fructose and lactose; colloidal substances such as animal proteins, e.g.
  • casein in particular sodium caseinate
  • vegetable proteins for example soy protein, pea protein, bean protein, lupine, zein, wheat protein, maize protein and rice protein
  • synthetic polymers for example polyethylene glycol, polyvinyl alcohol and in particular the Kollidon brands from BASF
  • optionally modified biopolymers for example Ligin, chitin, chitosan, polylactide and modified starches, for example octenyl succinate anhydride (OSA); Gums, eg gum acacia
  • Cellulose derivatives for example methyl cellulose, ethyl cellulose, (hydroxyethyl) methyl cellulose (HEMC), (hydroxypropyl) methyl cellulose (HPMC), carboxymethyl cellulose (CMC); Flours such as corn flour, wheat flour, rye flour, barley flour and rice flour.
  • carrier materials are carbohydrates, in particular the sugars mentioned above as binders, and starches, for example from corn, rice, potatoes, wheat and cassava; modified starches, for example octenyl succinate anhydride; Cellulose and microcrystalline cellulose; inorganic minerals or clay, for example clay, coal, diatomaceous earth, silica, tallow and kaolin; Semolina, for example wheat semolina, bran, for example wheat bran, the flours mentioned above as binders; Salts such as metal salts, in particular alkali and alkaline earth metal salts of organic acids, for example Mg, Ca, Zn, Na, K citrate, acetate, formate and hydrogen formates, inorganic salts, for example Mg, Ca, Zn , Na, K sulfates, carbonates, silicates or phosphates; Alkaline earth metal oxides such as CaO and MgO; inorganic buffering agents such as alkali metal hydrogen phosphates, in particular sodium
  • powdering agents or flow aids are diatomaceous earth, silica, e.g. the Sipernat brands from Degussa; Clay, coal, tallow and kaolin; the starches, modified starches, inorganic salts, salts of organic acids and buffering agents mentioned above as carrier materials; Cellulose and microcrystalline cellulose.
  • color pigments such as TiO 2 ; Bi-ozide; dispersant; Anti-foaming agent; Viscosity regulators; inorganic acids such as phosphoric acids, nitric acid, hydrochloric acid, sulfuric acid; organic acids such as saturated and unsaturated mono- and dicarboxylic acids, for example formic acid, acetic acid, propionic acid, butyric acid, valeric acid, palmitic acid, stearic acid, oxalic acid, malonic acid, succinic acid, glutaric acid, adipic acid, pimelic acid, maleic acid and fumaric acid; Alkalis such as alkali metal hydroxides, for example NaOH and KOH; antioxidants; Enzyme stabilizers; Enzyme inhibitors; adsorbates; fats; Fatty acids and oils.
  • inorganic acids such as phosphoric acids, nitric acid, hydrochloric acid, sulfuric acid
  • organic acids such as saturated and unsaturated mono- and di
  • the proportion of the abovementioned additives and, if appropriate, further additives, such as coating materials can vary widely depending on the special requirements of the particular metabolic product and depending on the properties of the additives used, for example in the range from 0.1 to 80% by weight, in particular in the range from 5 to 70% by weight and especially in the range from 10 to 60% by weight, in each case based on the total weight of the finished formulated product or in each case based on the total weight of the fully formulated product or mixture of substances.
  • Formulation auxiliaries can be added before, during or after the processing of the fermentation broth (also referred to as product formulation or solid design) and in particular during drying. Adding formulation aids before concentrating the fermentation broth remaining after step c) can be particularly advantageous in order to improve the processability of the substances or products to be processed.
  • the formulation auxiliaries can be added both to the by-product obtained in solid form and to a solution or suspension containing it, e.g. after step c) directly to the fermentation broth or to a solution or suspension obtained in the course of the work-up before the final drying step.
  • the auxiliary substances can e.g. are mixed into a suspension obtained by concentrating the fermentation broth remaining after step c); such a suspension can also be applied to a carrier material, e.g. by mixing in.
  • formulation aids are added e.g. when applying coatings or coatings / coating layers to dried particles. Additional aids can be added to the product both after drying and after a possible coating step.
  • the particles obtained by formulation processes can be dried to the desired residual moisture content by the drying processes described above.
  • All by-products obtained in solid form e.g. Particles, granules and extrudates can be coated with a coating, i.e. be covered with at least one further layer of material.
  • Coating takes place e.g. in mixers or fluidized beds in which the particles to be coated are whirled up or “fluidized” and then sprayed with the coating or coating material.
  • the coating material can be dry, for example as a powder, or in the form of a solution, dispersion, emulsion or suspension in a solvent, for example water, organic solvents and mixtures thereof, in particular in water. If present, the solvent is removed by evaporation during or after spraying onto the particles.
  • coating materials such as fats can also be applied as melts.
  • Coating materials that can be sprayed on as an aqueous dispersion or suspension are described, for example, in WO 03/059087.
  • These include in particular polyolefins such as polyethylene, polypropylene, polyethylene waxes, waxes, inorganic and organic salts, acronals, for example butyl acrylate-methyl acrylate copolymer, the styrofoam brands from BASF, for example based on styrene and butadiene, and hydrophobic substances such as described in WO 03/059086.
  • the solids content of the coating material is typically in the range from 0.1 to 20% by weight, in particular in the range from 0.2 to 10% by weight and especially in the ranging from 0.4 to 5 wt .-%, each based on the total weight of the formulated end product.
  • Coating materials that can be sprayed on as solutions are e.g. Polyethylene glycols, cellulose derivatives such as methyl cellulose, hydroxypropylmethyl cellulose and ethyl cellulose, polyvinyl alcohol, proteins such as gelatin, inorganic and organic salts, carbohydrates such as sugar, e.g. Glucose, lactose, fructose, sucrose and trehalose; Strengths and modified strengths.
  • the solids content of the coating material is typically in the range from 0.1 to 20% by weight, in particular in the range from 0.2 to 10% by weight and especially in the range from 0.4 to 5% by weight. %, each based on the total weight of the formulated end product.
  • Coating materials that can be sprayed on as a melt are described, for example, in DE 199 29 257 and WO 92/12645. These include in particular polyethylene glycols, synthetic fats and waxes, for example Polygenous WE ® from. BASF, natural fats such as animal fats, for example beeswax, and vegetable fats such as Candelila- wax, fatty acids, for example animal waxes, tallow fatty acid, palmitic acid, stearic acid , triglycerides, Edenor products, Vegeole products, Montanesterwachse such Luwax e ® from. BASF.
  • polyethylene glycols synthetic fats and waxes
  • synthetic fats and waxes for example Polygenous WE ® from. BASF
  • natural fats such as animal fats, for example beeswax
  • vegetable fats such as Candelila- wax
  • fatty acids for example animal waxes, tallow fatty acid, palmitic acid
  • the solids content of the coating material is typically in the range from 1 to 25% by weight, in particular in the range from 2 to 25% by weight and especially in the range from 3 to 20% by weight, in each case based on the total weight of the formulated end product.
  • whole or ground cereal grains preferably maize, wheat, barley, millet and / or rye, can be added to the by-product or the protein composition.
  • Another object of the invention is thus a protein composition from a sugar-based microbial fermentation carried out according to the invention for the production of a metabolite with at least 3 C atoms or with at least 2 C atoms and at least 1 N atom, which is obtainable as described above.
  • This protein composition usually contains protein material, i.e. Biomass from the fermentation, non-starchy components of the starch source, in particular fibers, and fermentation product (metabolite).
  • the protein composition essentially contains the following dry matter components:
  • e 0 to 40% by weight, in particular 0.5 to 30% by weight and especially 1 to 20% by weight of non-metabolized further constituents of the fermentation broth, in particular residues of sugar, starch, nutrient salts and / or buffer salts;
  • components a) to e) add up to 100% by weight of dry matter.
  • the expression “essentially” here means that the proportion of further constituents different from a) to e) is small, as a rule this proportion becomes 10% by weight and in particular 5% by weight, in each case based on the total dry mass of the protein composition, especially this proportion is less than 1% by weight, in particular about 0% by weight.
  • Biomass (component a)) includes in particular the proportion of crude protein in the protein composition. This proportion is usually at least 40% by weight and is in particular in the range from 40 to 90% by weight, especially in the range from 45 to 85% by weight and especially in the range from 50 to 80% by weight based on the total dry mass of the protein composition.
  • the protein compositions according to the invention usually contain one or more essential amino acids, in particular at least one amino acid selected from lysine, methionine, threonine and tryptophan.
  • the essential amino acids in particular those mentioned, are generally present in an amount which is increased by a factor of at least 1.5 compared to a conventional DDGS by-product which is obtained in fermentative bioethanol production. If the respective amino acid is contained in the protein composition, it generally has a lysine content of at least 1% by weight, in particular in the range from 1 to 5% by weight, and a methionine content of at least 0.8% by weight.
  • % in particular in the range from 0.8 to 5% by weight, a threonine content of at least 1.5% by weight, in particular in the range from 1.5 to 5% by weight and / or a Tryptophan content of at least 0.4% by weight, in particular in the range from 0.4 to 5% by weight, in each case based on the total dry mass of the protein composition.
  • the protein compositions according to the invention usually still contain a small proportion of water, frequently in the range from 0 to 25% by weight, in particular in the range from 0.5 to 15% by weight, especially in the range from 1 to 10% by weight and entirely especially in the range of 1 to 5 wt .-% water, each based on the total weight of the protein composition.
  • Another object of the invention is a method as described above, characterized in that
  • step a a partial amount of not more than 50% by weight is taken from the sugar-containing liquid medium obtained in step a), which contains the non-starchy solid constituents of the starch source, and with the remaining amount a fermentation according to b) to produce a first metabolic product (A) performs;
  • the non-starch-containing solid constituents of the starch source are completely or partially separated from this partial amount and this is used to carry out a fermentation according to b) to produce a second metabolic product (B) which is identical to or different from the metabolic product (A).
  • the non-starchy solid constituents are separated off in accordance with (ii) such that the solids content of the remaining portion of the sugar-containing liquid medium is at most 50% by weight, preferably at most 30% by weight, particularly preferably at most 10% % and very particularly preferably a maximum of 5% by weight.
  • microorganisms in the separate fermentation according to (iii) for which certain minimum requirements have to be met, e.g. regarding the oxygen transfer rate.
  • microorganisms used in the separate fermentation according to (iii) e.g. Bacillus species, preferably Bacillus subtilis.
  • the compounds produced by such microorganisms in the separate fermentation are in particular vitamins, cofactors and nutraceuticals, purine and pyrimidine bases, nucleosides and nucleotides, lipids, saturated and unsaturated fatty acids, aromatic compounds, proteins, carotenoids, especially vitamins, cofactors and Nutraceuticals, proteins and carotenoids and especially selected from riboflavin and calcium pantothenate.
  • this procedure enables the process according to the invention to be used advantageously even if the fine chemical produced is obtained as a solid during fermentation.
  • a preferred embodiment of this procedure relates to the parallel production of the same metabolic products (A) and (B) in two separate fermentations. This is particularly advantageous when there are different purity requirements for different applications of the same metabolic product.
  • the first metabolic product (A) e.g. an amino acid to be used as a feed additive, e.g. lysine
  • the same second metabolic product (B) e.g. the same amino acid to be used as a food additive, here e.g. lysine, using the prepared according to (ii) solids-depleted fermentation broth. Due to the complete The partial or partial removal of the non-starchy solid constituents can reduce the cleaning effort when working up the metabolic product, the area of application of which requires higher purity, for example as a food additive.
  • the metabolic product B produced by the microorganisms in the fermentation is riboflavin.
  • Analogous conditions and procedures as described for other carbon sources can be used here to carry out the fermentation, e.g. in WO 01/011052, DE 19840709, WO 98/29539, EP 1186664 and Fujioka, K .: New biotechnology for riboflavin (vitamin B2) and character of this riboflavin. Fragrance Journal (2003), 31 (3), 44-48.
  • a preferably large-volume fermentation for the production of metabolic products A e.g. of fine chemicals such as lysine, in accordance with process steps a) to c) according to the invention.
  • a part of the sugar-containing liquid medium obtained after step a) is removed and according to (ii) by conventional methods, e.g. Centrifugation or filtration, completely or partially freed from the solids.
  • the sugar-containing liquid medium obtained therefrom, essentially completely or partially freed from the solids is subjected to (ii) fermentation to produce a metabolic product B, e.g. Riboflavin supplied.
  • the solid stream separated according to (ii) is advantageously returned to the stream of the sugar-containing liquid medium of the large-volume fermentation.
  • the riboflavin-containing fermentation broth produced in this way according to (ii) can be worked up according to the analogous conditions and procedures described for other carbon sources, e.g. in DE 4037441, EP 464582, EP 438767 and DE 3819745. After lysing the cell mass, the crystalline riboflavin is preferably separated off by decanting. Other types of solids separation, e.g. Filtration are also possible. The riboflavin is then dried, preferably by means of spray and fluid bed dryers. Alternatively, the fermentation mixture containing riboflavin produced according to (ii) can be worked up according to analogous conditions and procedures, e.g. described in EP 1048668 and EP 730034. After pasteurization, the fermentation broth is centrifuged here and the remaining solid-containing fraction is treated with a mineral acid. The riboflavin formed is filtered from the aqueous acidic medium, washed if necessary and then dried.
  • the separated solids can be processed into a by-product in the course of the large-volume fermentation process operated in parallel, as already described above.
  • the metabolic product B produced by the microorganisms in the fermentation is pantothenic acid.
  • Conditions and procedures analogous to those described for other carbon sources can be used here to carry out the fermentation, for example in WO 01/021772.
  • the sugar-containing liquid medium pre-cleaned according to (ii), preferably essentially freed from the solids, is fed to a fermentation according to (ii) for the production of pantothenic acid.
  • the reduced viscosity compared to the liquid medium containing solids is particularly advantageous.
  • the separated solid stream is preferably returned to the stream of the sugar-containing liquid medium of the large-volume fermentation.
  • pantothenic acid-containing fermentation broth produced according to (ii) can be worked up according to analogous conditions and procedures as described for other carbon sources, e.g. in EP 1050219 and WO 01/83799. After pasteurizing the entire fermentation broth, the remaining solids are e.g. separated by centrifugation or filtration. The clarity of the solids separation is partially evaporated, calcium chloride optionally added and dried, in particular spray-dried.
  • the separated solids can be processed into a by-product in the course of the large-volume fermentation process operated in parallel, as already described above.
  • the metabolic product B produced by the microorganisms in the fermentation is polyhydroxyalkanoates.
  • Analogous conditions and procedures as described for other carbon sources can be used to carry out the fermentation, e.g. in S. Y. Lee, Plastic Bacteria? Progress and prospects for polyhydroxyalkanoate production in bacteria, Tibtech, Vol. 14, (1996), pp. 431-438.
  • the sugar-containing liquid medium pre-cleaned according to (ii), preferably substantially freed from the solids, is fed to a fermentation according to (ii) for the production of polyhydroxyalkanoates.
  • the separated solid stream is preferably returned to the stream of the sugar-containing liquid medium of the large-volume fermentation.
  • the fermentation broth produced according to (ii) and containing polyhydroxyalkanoate can be worked up according to the conditions and procedures analogous to those described for other carbon sources, for example in US 4310684 and EP 355307 Pasteurizing the entire fermentation broth, the remaining solids are separated, for example by centrifugation or filtration. The clarity of the solids removal is partially evaporated, calcium chloride optionally added and dried, in particular spray-dried. The further purification of the polyhydroxyalkanoates is carried out in a manner known per se, as described, for example, in US 4310684 or EP 355307.
  • the separated solids can be processed into a by-product in the course of the large-volume fermentation process operated in parallel, as already described above.
  • the regrind used below was produced as follows. Whole corn kernels were completely milled using a rotor mill. Three different subtleties were achieved using different racket mills, grinding tracks and sieve inserts. A sieve analysis of the ground material using a laboratory vibrating sieve (vibration analysis machine: Retsch Vibrotronic type VE1; sieving time 5 min; amplitude: 1.5 mm) gave the results listed in Table 1.
  • a further 400 g of the dry-ground corn meal (T71 / 03) were added over the course of 30 minutes, the temperature being raised to 91 ° C.
  • the reaction mixture was kept at this temperature for about 100 min.
  • a further 2.4 g of Termamyl 120L were added and the temperature was held for about 100 minutes.
  • the progress of the liquefaction was monitored during the period with the iodine-starch reaction.
  • the temperature was then raised to 100 ° C. and the reaction mixture was boiled for a further 20 min. At this point there was no longer any evidence of strength.
  • the reactor was cooled to 35 ° C.
  • the reaction mixture obtained from 11.1 a) was heated to 61 ° C. with constant stirring. Stirring was continued throughout the experiment. After adjusting the pH to 4.3 with H 2 SO 4 , 10.8 g (9.15 ml) of Dextrozyme GA (Novozymes A / S) were added. The temperature was maintained for about 3 hours, the progress of the reaction being monitored using glucose test sticks (S-Glucotest from Boehringer). The results are shown in Table 2 below.
  • the reaction mixture was then heated to 80 ° C. and then cooled. About 1180 g of liquid product with a density of about 1.2 kg / l and a dry matter content of about 53.7% by weight determined by means of an infrared dryer were obtained. The dry matter content (without water-soluble ingredients) was about 14% by weight after washing with water.
  • the proportion of glucose in the reaction mixture determined by HPLC was 380 g / l (cf. Table 2, sample No. 7).
  • a dry ground maize meal sample is liquefied in accordance with 11.1 a).
  • the reaction mixture obtained from II.2a) is heated to 61 ° C. with constant stirring. Stirring is continued throughout the experiment. After adjusting the pH to 4.3 with H 2 SO 4 , 10.8 g (9.15 ml) of Dextrozyme GA (Novozymes A / S) and 70 ⁇ l of phytase (700 units of phytase, Natuphyt Liquid 10000L from BASF Aktiengesellschaft) ) added. The temperature is held for about 3 hours, the progress of the reaction being monitored using glucose test sticks (S-Glucotest from Boehringer). The reaction mixture is then heated to 80 ° C. and then cooled. The product obtained is dried by means of an infrared dryer and washed with water. The proportion of glucose in the reaction mixture is determined by HPLC.
  • the addition of additional flour begins, initially 50 g of flour.
  • 0.13 ml of CaCl 2 stock solution is added to the mash in order to keep the Ca 2+ concentration at 70 ppm.
  • the temperature is kept constant at 85 ° C. Wait at least 10 minutes to ensure a complete reaction before adding another portion (50 g flour and 0.13 ml CaCl 2 stock solution).
  • 1.67 ml of Termamyl are added; then two further portions (50 g flour and 0.13 ml CaCl 2 stock solution each) are added. A dry matter content of 55% by weight is achieved.
  • the temperature is raised to 100 ° C. and the mash is boiled for 10 minutes.
  • a sample is taken and cooled to room temperature. After dilution of the sample with deionized water (about 1:10), a drop of concentrated Lugol's solution (mixture of 5 g I and 10 g Kl per liter) is added. A deep blue color indicates a content of remaining starch; browning occurs when the starch has been fully hydrolyzed. When the test shows a portion of starch remaining, the temperature is again lowered to 85 ° C and held constant. Another 1.67 ml of Termamyl are added until the iodine-starch reaction is negative.
  • the mixture tested negative for starch is then brought to 61 ° C. for the subsequent saccharification reaction.
  • the pH is adjusted to 4.3 by adding 50% sulfuric acid.
  • the pH is kept at this value in the course of the reaction.
  • the temperature is kept at 61 ° C.
  • 5.74 ml ( 1.5% by weight enzyme / dry matter)
  • Dextrozym GA Novozymes A / S
  • the reaction is allowed to proceed for an hour.
  • the mixture is heated to 85 ° C. to inactivate the enzyme.
  • the hot mixture is filled into sterile containers and stored after cooling at 4 ° C.
  • the addition of additional flour begins, initially 60 g of flour.
  • 0.13 ml of CaCl 2 stock solution is added to the mash in order to keep the Ca 2+ concentration at 70 ppm.
  • the temperature is kept constant at 85 ° C. Wait at least 10 minutes to ensure that the reaction is complete before adding a further portion (40 g of flour and 0.1 ml of CaCl 2 stock solution).
  • a further portion 40 g of flour and 0.1 ml of CaCl 2 stock solution
  • a dry matter content of 55% by weight is achieved.
  • the temperature is raised to 100 ° C.
  • the mixture tested negative for starch is then brought to 61 ° C. for the subsequent saccharification reaction.
  • the pH is adjusted to 4.3 by adding 50% sulfuric acid.
  • the pH is kept at this value in the course of the reaction.
  • the temperature is kept at 61 ° C.
  • 5.74 ml ( 1.5% by weight enzyme / dry matter)
  • Dextrozyme GA Novozymes A / S
  • the reaction is allowed to proceed for an hour.
  • the mixture is heated to 85 ° C. to inactivate the enzyme.
  • the hot mixture is filled into sterile containers and stored after cooling at 4 ° C.
  • an allelic exchange of the wild-type gene (lysC) coding for the enzyme aspartate kinase was carried out in C. glutamicum ATCC13032.
  • a nucleotide exchange was carried out in the lysC gene, so that the amino acid Thr at position 311 in the resulting protein was replaced by an III.
  • the oligonucleotide primers were used 5'-GAGAGAGACGCGTCCCAGTGGCTGAGACGCATC -3 '(SEQ ID NO: 1) and 5'-CTCTCTCTGTCGACGAATTCAATCTTACGGCCTG-3' (SEQ ID NO: 2) lysC using the Pfu-Turbo PCR system (Stratagene, USA) according to the manufacturer's PCR.
  • Chromosomal DNA from C. glutamicum ATCC 13032 was developed according to Tauch et al. (1995) Plasmid 33: 168-179 or Eikmanns et al.
  • the amplified fragment is flanked at its 5 'end by a Sall restriction site and at its 3' end by a Mlul restriction site. Before cloning, the amplified fragment was digested by these two restriction enzymes and purified using GFX TM PCR, DNA and Gel Band Purification Kit (Amersham Pharmacia, Freiburg).
  • the polynucleotide obtained was cloned via the Sall and Mlul restriction sections into pCLIK ⁇ MCS integratively SacB, hereinafter referred to as pCIS, (SEQ ID NO: 3) and transformed into E.coli XL-1 blue.
  • Selection for plasmid-bearing cells was achieved by plating on LB agar containing kanamycin (20 ⁇ g / ml) (Lennox, 1955, Virology, 1: 190). The plasmid was isolated and the expected nucleotide sequence was confirmed by sequencing.
  • the plasmid DNA was prepared using methods and materials from Quiagen. Sequencing reactions were carried out according to Sanger et al.
  • the sequencing reactions were separated and evaluated using ABI Prism 377 (PE Applied Biosystems, Rothstadt).
  • the plasmid obtained was named pCIS lysC (SEQ ID NO: 4). It comprises the following main areas:
  • the directed mutagenesis of the lysC gene from C. glutamicum was carried out using the Quick Change Kit (Stratagene, USA) according to the manufacturer's instructions.
  • the muta- genesis was carried out in the plasmid pCIS lysC (SEQ ID NO: 4).
  • the following oligonucleotide primers were synthesized for the exchange of thr 311 to 311 ile using the Quickchange method (Stratagene): 5'-CGGCACCACCGACATCATCTTCACCTGCCCTCGTTCCG -3 '(SEQ ID NO: 5)
  • the use of these oligonucleotide primers in the Quickchange reaction leads to an exchange of the nucleotide in position 932 (from C to T) in the lysC gene (SEQ ID NO: 7).
  • the resulting amino acid exchange Thr3111le in the lysC gene was confirmed by a sequencing reaction after transformation into E.coli XL1-blue and plasmid preparation.
  • the plasmid was named pCIS lysC thr311 ile (SEQ ID NO: 8). It comprises the following main areas:
  • the plasmid pCIS lysC thr311 ile was transformed into C. glutamicum ATCC13032 by means of electroporation, as described in Liebl et al., FEMS Microbiology Letters 53: 299-303 (1989). Modifications to the protocol are described in DE 10046870.
  • the chromosomal arrangement of the lysC locus of individual transformants was determined using standard methods by Southern blot and hybridization, as in Sambrook et al., Molecular Cloning. A Laboratory Manual, Cold Spring Harbor (1989). This ensured that the transformants are those which have integrated the transformed plasmid by homologous recombination at the lysC locus. After growth of such colonies overnight in media containing no antibiotic, the cells are plated on a sucrose CM agar medium (10% sucrose) and incubated for 24 hours at 30 ° C.
  • the sacB gene containing sacB in the vector pCIS lysC thr311 ile converts sucrose into a toxic product, only those colonies can grow that the sacB gene through a second homologous recombination step between the wild-type gene lysC and have deleted the mutated gene lysC thr311ile. During homologous recombination, either the wild-type gene or the mutated gene can be deleted together with the sacB gene. If the sacB gene is removed together with the wild-type gene, a mutant transformant results.
  • Example 1 Enzymatic starch liquefaction and saccharification
  • Example 11.1 Two maize meal hydrolyzates obtained according to Example 11.1 were used in shake flask experiments using Corynebacterlum glutamicum (flasks 4-9). In addition, a wheat flour hydrolyzate (flask 1 -3) prepared in analogy to Example 11.1 was used in parallel.
  • CM agar composition: see Table 4; 20 min at 121 ° C.
  • the cells are incubated at 30 ° C. for 48 h.
  • the cells are then scraped off the plates and resuspended in saline.
  • 25 ml of the medium see Table 5
  • the samples are then incubated at 200 rpm and 30 ° C. in a humidified shaking cabinet (85% relative atmospheric humidity) for 48 hours.
  • the concentration of the lysine in the media is determined by means of HPLC. In all cases, approximately equal amounts of lysine were produced.
  • a maize meal hydrolyzate obtained according to Example II.3a was used in shake flask tests using Corynebacterlum glutamicum (ATCC13032 lysC fbr ) (flasks 1 + 2).
  • Corynebacterlum glutamicum ATCC13032 lysC fbr
  • a wheat flour hydrolyzate flask 3 + 4
  • rye flour hydrolyzate flask 5 + 6
  • compositions of the piston media 1 to 6 are listed in Table 8. An appropriate amount of glucose solution was used in the control medium instead of flour hydrolyzate.
  • lysine was produced in comparable amounts on the order of approximately 10 to 12 g / l, corresponding to the yield achieved in a standard fermentation with glucose nutrient solution. ⁇
  • the solids are usually separated from the fermentation broth by centrifugation in a first step.
  • centrifugation filtration processes such as membrane filtration can also be used.
  • the solids-free fermentation broth is then acidified (for example with sulfuric acid), as a result of which lysine is present in one or two protonated forms.
  • This acidified broth is then passed through a cation exchanger so that the lysine binds to the ion exchanger. After rinsing with water, ammonia water is then passed over the ion exchanger to elute the lysine.
  • a maize meal hydrolyzate obtained according to Example II.3a was used in shake flask tests (flasks 1-3). Bacillus PA824 was used as the panthothenate-producing strain (detailed description in WO 02/061108). In addition, a wheat flour hydrolyzate (flask 4-6) and rye flour hydrolyzate (flask 7-9) prepared in analogy to Example II.3 were used in parallel. 4.1) Preparation of the inoculum
  • pantothenic acid was produced in comparable amounts in the order of about 1.5 to 2 g / l, corresponding to the yield achieved in a standard fermentation with glucose nutrient solution.
  • Refurbishing the product can e.g. as described in WO 02/24001, WO 02/072857 and WO 05/028659.
  • a maize meal hydrolyzate obtained according to Example II.3a was used in shake flask tests using Aspergillus niger (flasks 1-3).
  • a wheat flour hydrolyzate (flask 4-6) and rye flour hydrolyzate (flask 7-9) prepared in analogy to Example II.3 were used in parallel.
  • An Aspergillus niger phytase production strain with 6 copies of the Aspergillus ficuum phyA gene under the control of the glaA promoter was generated analogously to the production of NP505-7 described in detail in WO98 / 46772.
  • a strain with 3 modified glaA ampicons (analog ISO505) was used, but without integrated phyA expression cassettes.
  • compositions of the piston media 1 to 9 are listed in Table 14. An appropriate amount of glucose solution was used in the control medium instead of flour hydrolyzate. Table 14: Piston media
  • the flasks were incubated for 6 days at 34 ° C and with agitation (170 rpm) in a humidified shaker. After the fermentation was stopped, the phytase activity was determined using an assay. After termination of the fermentation, the phytase activity with phytic acid as substrate and at a suitable phytase activity level (standard: 0.6 U / ml) in 250 mM acetic acid / sodium acetate / Tween 20 (0.1% by weight), pH 5.5 Buffer determined. The assay has been standardized for use in microtiter plates (MTP).
  • MTP microtiter plates
  • the product can be worked up as described in WO 98/55599.
  • a maize meal hydrolyzate obtained according to Example II.3a was used in shake flask tests using Ashbya gossypii (flasks 1-4).
  • a wheat flour hydrolyzate (flask 5-8) and rye flour hydrolyzate (flask 9-12) prepared in analogy to Example II.3 were used in parallel. 6.1) strain
  • the riboflavin-producing strain used is Ashbya gossypii ATCC 10895 (see also Schmidt G, et al. Inhibition of purified isocitrate lyase identified itaconate and oxalate as potential antimetabolites for the riboflavin overproducer Ashbya gossypii. Microbiology 142: 411 -417, 1996 ).
  • the product can be worked up as described in EP 00345717.
  • a maize meal hydrolyzate obtained according to Example II.3a was used in shake flask tests using Corynebacterlum glutamicum (flasks 1-3).
  • a wheat flour hydrolyzate (flask 4-6) and rye flour hydrolyzate (flask 7-9) prepared in analogy to Example II.3 were used in parallel.
  • a maize meal hydrolyzate obtained according to Example II.3a was used in shake flask experiments using Bacterium 130Z. 8.1) strain
  • Bacterium 130Z (ATCC No. 55618) was used as the succinate-producing strain. 8.2) Preparation of the fermentation broth
  • the composition of the medium is listed in Table 23 (cf. US 5,504,004). A corresponding amount of glucose solution was used in the control medium instead of flour hydrolyzate (final concentration glucose: 100 g / l). Table 23: Medium *
  • a maize flour hydrolyzate obtained according to Example II.3a is used in shake flask experiments using Escherichia coli (flask 1-3). Similarly, a wheat flour hydrolyzate (flask 4-6) and rye flour hydrolyzate (flask 7-9) produced in the same way as in Example II.3 are used in parallel.
  • Escherichia co / strains which produce L-threonine are known to the person skilled in the art.
  • the production of such strains is e.g. in EP 1013765 A1, EP 1016710 A2, US 5,538,873.
  • compositions of the piston media 1 to 9 are listed in Table 25.
  • a corresponding amount of glucose solution is used in the control medium instead of flour hydrolyzate.
  • the flasks are incubated at 30 ° C and with agitation (200 rpm) in a humidified shaker until the glucose is used up.
  • the L-threonine content can be determined by reversed-phase HPLC as described by Lindroth et al., Analytical Chemistry 51: 1167-1174, 1979.
  • the fermentation broth can then be harvested and the L-threonine contained in the fermentation broth can be isolated, purified or otherwise processed, e.g. in US 5,538,873 and by Okamoto et al., Bioscience, Biotechnology and Biochemistry 61 (11), 1877-1882, 1997.
  • a cassava flour hydrolyzate obtained analogously to Example II.3 was used in shake flask experiments using the lysine-producing strain of Corynebacterlum glutamicum described under 11.2) (flask 1 -4).
  • the flour used had the following size distribution: 45% ⁇ 100 ⁇ m, 56% ⁇ 200 ⁇ m, 79% ⁇ 630 ⁇ m.
  • the viscosity of the suspension was already relatively high at the beginning of the liquefaction step, so that initially cassava flour was used in an amount corresponding to a dry matter content of 35% by weight. There was a correspondingly increased addition of flour in order to ultimately achieve a dry matter content of 55% by weight.
  • the viscosity of the suspension remained relatively high throughout the liquefaction and saccharification.
  • the cassava flour tended to clump; the lumps only partially dissolved again in the further course. Existing lumps turned deep blue in the iodine starch test after a few minutes; this indicates that the clumped starch could not be fully implemented despite multiple boilings and longer waiting times. 11.1) strain
  • CM + CaAc agar composition: see Table 26; 20 min at 121 ° C.
  • the cells were incubated at 30 ° C. for 24 h.
  • the cells were then scraped off the plates and resuspended in saline.
  • the composition of the piston medium is shown in Table 27. An appropriate amount of glucose solution was used in the control medium instead of flour hydrolyzate.
  • lysine was produced in comparable amounts in the order of approximately 10 to 14 g / l, corresponding to the yield achieved in a standard fermentation with glucose nutrient solution.
  • a partially saccharified corn flour hydrolyzate was used in shake flask experiments using Aspergillus niger.
  • Dextrozyme GA Novozymes A / S
  • Example 5.1 The strain used in Example 5.1 was used.
  • the inoculum was prepared as described in Example 5.2).
  • the compositions of the piston medium listed in Table 29 were used to prepare the fermentation broth. Two flasks were made from each sample.
  • a partially saccharified corn flour hydrolyzate was used in shake flask experiments using Corynebacterlum glutamicum.
  • Dextrozyme GA Novozymes A / S
  • Example 3 The strain used in Example 3 was used.
  • the inoculum was produced as described in Example 3.1).
  • compositions of the flask medium listed in Table 31 were used to prepare the fermentation broth. Three flasks were made from each sample.
  • Example 14 In a fermentation for the production of lysine carried out analogously to example 3, after depletion of the lysine from the fermentation broth in step c), a protein composition was obtained as the dried fermentation residue.
  • Table 33 shows essential components of the composition with their weight fractions and compared with a conventional DDGS composition.
  • Table 33 Analysis results based on dry matter in% by weight **

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Abstract

Procédé de production d'au moins un métabolite microbien ayant au moins 3 atomes de C ou au moins 2 atomes de C et au moins un atome de N par fermentation microbienne à base de sucre. Ledit procédé consiste (a) à produire un milieu liquide saccharifère ayant une teneur en monosaccharide supérieure à 20 % en poids à partir d'une source d'amidon, ledit milieu liquide saccharifère comprenant également des constituants solides ne contenant pas d'amidon et provenant de la source d'amidon, (b) à faire fermenter le milieu liquide saccharifère en vue de la production du (des) métabolite(s) et (c) à séparer ou à isoler au moins un métabolite du bouillon de fermentation. Selon ledit procédé, une souche de micro-organismes produisant le (les) métabolite(s) souhaité(s) est cultivée avec le milieu liquide saccharifère que l'on obtient (a1) en moulant la source d'amidon et (a2) en liquéfiant la matière moulue dans un liquide aqueux en présence d'au moins une enzyme liquéfiant l'amidon, puis en saccharifiant le liquide obtenu à l'aide d'au moins une enzyme de saccharification, au moins une quantité partielle de la matière moulue étant liquéfiée par ajout contenu et ponctuel de ladite matière au liquide aqueux.
EP05754405A 2004-05-28 2005-05-27 Production de produits chimiques fins par fermentation Withdrawn EP1753868A2 (fr)

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MX291201B (es) 2011-10-20
ZA200610738B (fr) 2008-04-30
PH12006502292B1 (en) 2014-02-13
JP2008500823A (ja) 2008-01-17
EA015492B1 (ru) 2011-08-30
US20090162892A1 (en) 2009-06-25
AU2005248061A1 (en) 2005-12-08
KR101317064B1 (ko) 2013-10-16
JP4659825B2 (ja) 2011-03-30
AR049122A1 (es) 2006-06-28
US9109244B2 (en) 2015-08-18
UA89785C2 (uk) 2010-03-10
CA2566475A1 (fr) 2005-12-08
WO2005116228A2 (fr) 2005-12-08
EA015492B9 (ru) 2012-02-28
TW200613557A (en) 2006-05-01
EA200602068A1 (ru) 2007-06-29
KR20120101724A (ko) 2012-09-14
KR101479583B1 (ko) 2015-01-06
AU2005248061B2 (en) 2010-02-11
WO2005116228A3 (fr) 2006-05-11
KR20070021226A (ko) 2007-02-22
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